Optimization of GFP introduction into HL-60 cells with a combination of two different rectangular pulses

Electroporation is a method to introduce genes into cells by using electric pulses. To achieve a high efficiency of electroporation, it is very important to choose appropriate parameters of electric pulses. In this study, transformation efficiencies of HL-60 cells (human promyelocytic leukemia cells) were investigated by using a combination of two different rectangular pulses. The pulses consisted of a short pulse with higher voltage to create pores on cell membrane as 1st pulse and a longer pulse with lower voltage to move a GFP (green fluorescent protein) plasmid DNA into HL-60 cells as 2nd pulse. To determine the 1st pulse, 200 ns pulse (2 kV, 20 mJ) which was seldom used in ordinary electroporators and 10 μs pulse (300 V, 22.5 mJ) were tested and compared. The results by using combination of each pulse and 100 ms pulse (60 V, 9.0 J) as 2nd pulse showed that transformation efficiency by using 200 ns pulse as 1st pulse was better than that by 10 μs pulse. To evaluate the 2nd pulse effects, various types of the 2nd pulse parameter from 10 μs to 1 s for pulse duration and from 4 V to 400 V for voltage were tested. The outcomes of transformation efficiencies were posted in a graph with an electric field on the perpendicular axis and an input energy on the horizontal axis. The graph was roughly divided into three areas, which were “no effect”, “too much damage”, and “good introduction” by the transformation efficiency. This result indicated that better pulse parameters for the 2nd pulse were easily found by using this graph.